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Molecular Mechanism of Functional Expression of the Chaperonin

伴侣蛋白功能表达的分子机制

基本信息

批准号：
10480177
负责人：
KUWAJIMA Kunihiro
金额：
$ 8.38万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

The objective of the present study is to elucidate the molecular mechanism of functional expression of the Escherichia coli chaperonin GroEL/ES. For this purpose, we investigated the interactions of the chaperonin with folding intermediates of three globular proteins, α-lactalbumin (αLA), staphylococcal nuclease (SNase) and green fluorescent protein (GFP), which have very different physical properties. We also investigated physicochemically the interactions of GroEL with the nucleotides (ATP and ADP) that are indispensable for the chaperonin function and the analogs of the nucleotides (ATPγS and AMP-PNP). The following results were obtained.(1) We used an SNase mutant, in which the refolding kinetics were significantly simplified, and apo-αLA that showed a simple single relaxation kinetics of refolding, as model target proteins of GroEL. We studied the effect of the chaperonin on the refolding kinetics of these target proteins by stopped-flow fluorescence spectroscopy. Especially for α … More LA, we succeeded the quantitative analysis of the reaction curves by computer simulations. When we added a nucleotide in the absence of GroES, only ATP was effective for reducing the affinity of GroEL for the target protein. However, when GroES was present, not only ATP but also the ATP analogs were found to effectively reduce the GroEL affinity for the target protein.(2) We constructed E. coli expression systems for GFP and its Cycle3 mutant and studied their in vitro refolding reactions by fluorescence spectroscopy. These proteins can also be used as model target proteins of the chaperonin.(3) We studied the interactions of GroEL with ADP and the ATP analog by titration calorimetry and fluorescence spectroscopy. In the latter method, we used the fluorescence intensity change of pyrenyl GroEL to monitor the interactions. We found that these nucleotides bound to GroEL in a non-cooperative manner and that there were two kinds of the binding sites with different affinities. Only ATP induced a cooperative fluorescence change of pyrenyl GroEL., suggesting that ATP hydrolysis was required for the cooperative change. From this results together with the above results in(1), the ATP hydrolysis and the resultant cooperative change are expected to by required for releasing the target protein form GroEL. Less

本研究的目的是阐明大肠杆菌伴侣蛋白GroEL/ES功能表达的分子机制。为此，我们研究了分子伴侣蛋白与三种球形蛋白质（α-乳白蛋白（αLA）、葡萄球菌核酸酶（SNase）和绿色荧光蛋白（GFP））折叠中间体的相互作用。我们还研究了GroEL与伴侣蛋白功能所必需的核苷酸（ATP和ADP）及其类似物（ATPγS和AMP-PNP）的相互作用。获得了以下结果。(1)我们使用SNase突变体和apo-αLA作为GroEL的模型靶蛋白，其中SNase突变体的重折叠动力学显著简化，apo-αLA显示简单的单松弛重折叠动力学。我们通过停流荧光光谱研究了分子伴侣对这些靶蛋白复性动力学的影响。尤其是α ...更多信息通过计算机模拟，成功地对反应曲线进行了定量分析。当我们在没有GroES的情况下添加核苷酸时，只有ATP有效降低GroEL对靶蛋白的亲和力。然而，当GroES存在时，发现不仅ATP而且ATP类似物都有效地降低了GroEL对靶蛋白的亲和力。(2)我们构建了E.大肠杆菌表达系统，并通过荧光光谱研究了GFP及其Cycle 3突变体的体外复性反应。这些蛋白质也可用作伴侣蛋白的模型靶蛋白。(3)我们用滴定量热法和荧光光谱法研究了GroEL与ADP和ATP类似物的相互作用。在后一种方法中，我们利用芘基GroEL的荧光强度变化来监测相互作用。我们发现这些核苷酸与GroEL以非合作方式结合，并且存在两种亲和力不同的结合位点。只有ATP诱导芘基GroEL.的协同荧光变化，表明ATP水解是协同变化所必需的。根据该结果和上述（1）的结果，可以推测，ATP水解和由此产生的协同变化是从GroEL释放靶蛋白质所必需的。少